Radcot

Determines bacterial genomes from DNA reads then shows gene expression differences from RNA reads

This is a monolithic program that combines the three steps (using the programs centrifuge, bowtie2, samtools, htseq-count, deseq2, and numerous python and R libraries)

The idea is that this one program combines all three steps so you do not have to run them separately.

Table of Contents

• Radcot
  • centrifuge-patric - Radcot Part One - 1st step
  • bowtie-samtools - Radcot Part Two - 2nd step
  • count-deseq - Radcot Part Three - 3rd step
• Installation / Quick start
• Example run
  • Getting the example data
  • Running it
• References
• Detailed developer notes are here as a google doc

Created by gh-md-toc

These are the individual steps if you would like to install / run them separately. CAUTION: I do not guarantee that these are up-to-date with the full Radcot

centrifuge-patric - Radcot Part One - 1st step

• Identify bacterial species from a metagenomic sample with DNA alignment
• Download genomes and annotations of said species

bowtie-samtools - Radcot Part Two - 2nd step

• Align RNA reads to abundanct bacterial genomes

count-deseq - Radcot Part Three - 3rd step

• Get counts of RNA alignments to transcript-producing genes (CDS)
• Run Deseq2 guided by the experiment setup file (metadata.txt) that will output graphs / tables of differentially expressed genes

Installation / Quick start:

1. Use https://www.imicrobe.us/#/apps to access the app with your cyverse login OR
2. git clone https://github.com/hurwitzlab/radcot on a linux system where you have admin rights
3. Install singularity
4. Build the singularity image in singularity/ by executing make img
5. Upload all the files to an HPC with a slurm scheduler¹
6. Run the program with sbatch run.sh [arguments]

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¹This can be adapted to run on other batch-scheduled high-performance computer systems but this has not been tested.

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Example run:

Getting the example data

1. Have iRODS tools installed on your HPC
2. Get the example data with iget -r /iplant/home/shared/imicrobe/example_data/radcot-data * If using iMicrobe, this will be your input directory and you shouldn't need to download
3. To check you have the data, make sure each DNA and RNA read file matches those found in metadata_template.txt

Running it

4. Move the radcot.img into /stampede
5. Issue make test
6. Detail of expected output can be found in the google doc at the bottom of the page but briefly: * You should see centrifugue reports, genomes, bam files, count files and finally deseq graphs and tables in the output

References:

• http://singularity.lbl.gov/all-releases
• https://ccb.jhu.edu/software/centrifuge/
• https://docs.patricbrc.org/cli_tutorial/index.html
• http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
• http://htseq.readthedocs.io/en/master/count.html
• https://github.com/PF2-pasteur-fr/SARTools

Detailed developer notes are here as a google doc

Thank you to @kyclark and @bhurwitz33 for helpful comments and suggestions.